U.S. patent application number 10/532446 was filed with the patent office on 2006-06-08 for method for the selective and quantitative functionalization of immunoglobulin fab fragments, conjugate compounds obtained with the same and compositions thereof.
This patent application is currently assigned to Bracco Imaging S.P.A.. Invention is credited to Christoph De Haen, Federico Maisano.
Application Number | 20060120959 10/532446 |
Document ID | / |
Family ID | 32116242 |
Filed Date | 2006-06-08 |
United States Patent
Application |
20060120959 |
Kind Code |
A1 |
De Haen; Christoph ; et
al. |
June 8, 2006 |
Method for the selective and quantitative functionalization of
immunoglobulin fab fragments, conjugate compounds obtained with the
same and compositions thereof
Abstract
The invention provides chemical conjugates between an
immunoglobulin Fab fragment and molecular entities imparting
diagnostic or therapeutic utility, whereby the only sites of
conjugation on the Fab fragment are one or both of the sulfhydryl
groups deriving from the selective and quantitative reduction of
the inter-chain disulfide bond of said Fab fragment and whereby
said molecular entities imparting diagnostic or therapeutic utility
have at least one free sulfhydryl-reactive group, characterized in
that the conjugation stoichiometric molar ratio molecular entity to
Fab fragment is in the range from 0.95 to 1.05 or in the range from
1.95 to 2.05. The invention also provides a process for preparing
said conjugates and pharmaceutical compositions thereof.
Inventors: |
De Haen; Christoph;
(Peschiera Borromeo, IT) ; Maisano; Federico;
(Lodi, IT) |
Correspondence
Address: |
BRACCO RESEARCH USA INC.
305- COLLEGE ROAD EAST
PRINCETON
NJ
08540
US
|
Assignee: |
Bracco Imaging S.P.A.
Via Egidio Folli, 50
Milano
IT
20134
|
Family ID: |
32116242 |
Appl. No.: |
10/532446 |
Filed: |
November 10, 2003 |
PCT Filed: |
November 10, 2003 |
PCT NO: |
PCT/EP03/12514 |
371 Date: |
April 22, 2005 |
Current U.S.
Class: |
424/1.49 ;
424/178.1; 530/391.1; 548/546 |
Current CPC
Class: |
A61K 47/6887 20170801;
A61K 51/1093 20130101; B82Y 5/00 20130101; A61K 47/6897
20170801 |
Class at
Publication: |
424/001.49 ;
424/178.1; 530/391.1; 548/546 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/46 20060101 C07K016/46; C07D 207/40 20060101
C07D207/40; A61K 51/00 20060101 A61K051/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 13, 2002 |
EP |
02025315.9 |
Claims
1. A chemical conjugate between an immunoglobulin Fab fragment and
molecular entities imparting diagnostic or therapeutic utility,
whereby the only sites of conjugation on the Fab fragment are one
or both of the sulfhydryl groups deriving from the selective and
quantitative reduction of the inter-chain disulfide bond of said
Fab fragment and whereby said molecular entities imparting
diagnostic or therapeutic utility have at least one free
sulfhydryl-reactive group, characterized in that the conjugation
stoichiometric molar ratio of molecular entity to Fab fragment is
in the range from 0.95 to 1.05 or in the range from 1.95 to
2.05.
2. A conjugate according to claim 1, wherein the first of said
sulfhydryl groups deriving from the selective and quantitative
reduction of the inter-chain disulfide bond is quantitatively
functionalized by reaction with one of said molecular entities
imparting diagnostic or therapeutic utility.
3. A conjugate according to claim 2, wherein the second of said
sulfhydryl groups deriving from the selective and quantitative
reduction of the inter-chain disulfide bond is quantitatively
functionalized by reaction with a second of said molecular entities
imparting diagnostic or therapeutic utility, said second molecular
entity being different or identical to the first one.
4. A conjugate according to claim 1, wherein both of said
sulfhydryl groups deriving from the selective and quantitative
reduction of the inter-chain disulfide bond are quantitatively
symmetrically functionalized by reaction with a stoichiometric
excess of one of said molecular entities imparting diagnostic or
therapeutic utility.
5. A conjugate according to claim 1, wherein one of said sulfhydryl
groups deriving from the selective and quantitative reduction of
the inter-chain disulfide bond is chemically modified by reaction
with a chemical moiety non-imparting diagnostic or therapeutic
utility, said chemical moiety being preferably selected from the
group consisting of protective groups of the thiol groups of small
alkylating and arylating agents.
6. A conjugate according to claim 1, wherein said molecular
entities imparting diagnostic or therapeutic utility comprise
derivatives of chelating agents for, or chelates of, radionuclides,
paramagnetic metal ions or luminescent metal ions, a chromophoric
fluorescent or a phosphorescent molecule, a biotin molecule, a
hapten recognized by a distinct antibody or fragment thereof, an
avidin or streptavidin molecule, a therapeutic drug, a lipophilic
chain bearing molecular entity incorporated into liposomes,
phospholipid-stabilized microbubbles, triglyceride- or
polymer-based microspheres, microballoons which carry the
diagnostic or therapeutic agent.
7. A conjugate according to claim 6, wherein said molecular
entities further comprise one or more functional groups which may
be used, as such or after deprotection or after chemical
modification, as targets for the selective attachment of a second
Fab fragment, equal or different from the first one, or of a second
molecular entity imparting diagnostic or therapeutic utility.
8. A conjugate according to any one of claims 1 to 6, wherein said
sulfhydryl reactive groups comprise iodoacetyl, bromoacetyl, vinyl,
maleimido groups or polyfluorobenzene or dinitrofluorobenzene
derivatives.
9. A conjugate according to any one of claims 1 to 7, wherein the
Fab fragment is a recombinant Fab.
10. A process for the preparation of the conjugate of claim 1,
comprising: a) the selective and quantitative reduction of the
inter-chain disulfide bond of a Fab fragment to give two free
sulfhydryl groups; b) the quantitative functionalization of one or
both of the sulfhydryl groups from step a) with molecular entities
having at least one free sulfhydryl-reactive group and imparting
diagnostic or therapeutic utility, to give mono- or diconjugate
compounds, said diconjugates deriving from either symmetric or
asymmetric functionalization of the sulfhydryl groups.
11. The process of claim 10, wherein said selective and
quantitative reduction of the inter-chain disulfide bond is
performed with a phosphine, preferably tributylphosphine and
tris-(carboxyethyl)-phosphine.
12. The process of claim 11, wherein the reducing agent is
tris-(carboxyethyl)-phosphine.
13. The process of claim 11, wherein the reduction is carried out
mixing the reacting species under buffered conditions giving a
final buffered aqueous reaction solution having the following
characteristics: Fab concentration: 1-100 .mu.m; Phosphine
concentration: 0.1-10 mm; pH of the buffered solution: 4-8;
reaction time: 5-180 min; reaction temperature: 4-45.degree. C.
14. The process of claim 13, wherein the conditions are the
following: Fab concentration: 1.5-10 .mu.M or 1-5 .mu.M; Phosphine
concentration: 0.5-5 mM; pH of the buffered solution: 5-7; reaction
time: 25-70 min; reaction temperature: 25-40.degree. C.
15. The process of claim 10, wherein said quantitative
functionalization of step b) is performed immediately at the end of
the reduction step a), in the same reaction medium, by adding a
buffered aqueous solution of the conjugating molecular entity,
without purifying the reduced Fab fragment.
16. The process of claim 15, wherein the final buffered aqueous
reaction solution has the following characteristics: Fab
concentration: 2-5 .mu.M; Phosphine concentration: 0.5-5 mM;
conjugating moiety concentration: 0.1-100 mM; pH of the buffered
solution: 5-7; reaction time: .gtoreq.30 min; reaction temperature:
4-45.degree. C. or 20-40.degree. C.
17.
N.sup.2,N.sup.2-bis[2-[bis(carboxymethyl)amino]ethyl]-N.sup.6-[4-(2,5-
-dioxo-1H-pyrrol-1-oxobutyl]-L-lysine as intermediate compound for
the preparation of conjugates of claim 1.
18. Pharmaceutical compositions containing as active ingredients
the conjugate compounds of claim 1.
19. Compositions according to claim 18, wherein said conjugate
compounds are formulated in the form of suspensions, solutions,
emulsions for parenteral administration, lyophilizates to be
reconstituted before use.
20. Compositions according to claim 18, wherein the dose of the
active ingredient ranges from 0.1 to 10 mg of conjugate per single
administration.
21. Compositions according to claim 18, wherein the dose of the
active ingredient ranges from 10 to 500 mg of conjugate per single
administration.
22. Compositions according to claim 18, for use in analytical
immunochemical tests in vitro.
Description
FIELD OF THE INVENTION
[0001] This invention relates to conjugates of immunoglobulin Fab
fragments (Fab), in which said Fab have been quantitatively and
selectively functionalized only at predetermined specific desired
sites of the molecule.
[0002] The invention also relates to a method for obtaining said
selective and quantitative functionalization, as well as to
pharmaceutical compositions comprising said conjugates.
BACKGROUND OF THE INVENTION
[0003] Monoclonal antibodies (mAb) are proteins with the well-known
capability to localise both in vitro and in vivo on cells or on
tissues which expose the antigen to which they are specific. This
property is maintained in some of their well-known proteolytic
fragments, e.g. Fab, Fab' and F(ab').sub.2. In particular,
immunoglobulin Fab fragments (hereafter also in the plural simply
called Fab) maintain this property.
[0004] It is well known that diagnostic or therapeutic molecules of
different type, or precursors thereof, may be covalently linked to
a mAb or its fragments. Those conjugates in which the linked
diagnostic or therapeutic molecule does not interfere with the
capability of binding to the target antigen are able to transport
and thus target the molecule to antigen-bearing cells and tissues,
where it can exert its intended purpose, such as, for example,
diagnostic signals production or therapeutic cell killing.
[0005] Fab fragments are of particular interest as diagnostic or
therapeutic agents, since they are smaller than intact
immunoglobulins or some of their other fragments, e.g.
F(ab').sub.2. Smallness increases their rate of passage from the
blood to the tissue interstitium, where many of them find their
target. It also increases their diffusivity in the tissue
interstitium, and thereby it facilitates and accelerates their
arrival at the target site and the disappearance of unbound
molecules from said site. Moreover it increases their rate of
excretion, thus favouring the reduction of non-specific background
effects.
[0006] Only minimally larger than Fab fragments are Fab' fragments.
These are obtained from F(ab').sub.2 fragments by reduction of the
disulfide bridge linking the two heavy chains, and need to be
stabilized by chemical modification of the free sulfhydryl
groups.
[0007] Many methods for conjugating suitable diagnostic or
therapeutic molecules to mAb and their various fragments have
already been described. Typically the conjugating molecule modifies
the mAb or its fragments at various sites, including some that
interfere with binding to antigen. Major loss of binding to antigen
can often be achieved by a low stoichiometric ratio of conjugating
molecule to protein. Especially for radiodiagnostic purposes
stoichiometric ratios substantially below one are sometimes,
although not always, acceptable. When they are acceptable, the
explanation is found on the one hand in the elevated number of
antigenic sites relative to the number of mAb or its fragments
necessary for satisfactory signal generation, and on the other hand
in the fact that elevated concentrations of the mAb or its fragment
have no detrimental pharmacological activity. In these particular
cases excesses of unlabeled mAb or its fragments do not
significantly interfere with binding of their radiolabeled
conjugates and, from a signal-generating standpoint, they are
silent.
[0008] In contrast, when the number of antigenic sites is very low,
or when the mAb or its fragments have detrimental pharmacological
activities, it is highly preferable that most or all mAb or
fragments are radiolabeled.
[0009] The requirement for an elevated fraction, preferably the
totality, of mAb or fragments being radiolabeled is particularly
pronounced in the case of radiotherapy. In this case the number of
antigenic binding sites is almost always limiting therapeutic
efficacy. The same requirement holds when the mAb or its fragment
has detrimental pharmacological activity, which needs to be
contained. In these cases occupation of any sites by unlabeled mAb
or fragment is to be avoided.
[0010] Elevated stoichiometric ratios of conjugating molecules to
protein can easily be achieved for example by chemical modification
of free amino groups (amino-terminal .alpha.-amino groups and the
.di-elect cons.-amino groups of lysines) or of free carboxyl groups
(carboxy-terminal .alpha.-carboxy groups and the .gamma.-,
respectively .delta.-carboxy groups, of aspartic and glutamic
acid). Unfortunately, most of the times this is accompanied by the
creation of a substantial fraction of conjugates that binds no more
or poorly to antigen. When radiolabeled, such conjugates add noise
instead of signal to diagnostic procedures and add radiation load
without concomitant therapeutic benefits to therapeutic regimens.
Moreover, the described chemical conjugation methods are
non-specific for selected sites of the protein and consequently are
not useful for obtaining final products in which the numbers and/or
the types of the sites of conjugation on the protein or protein
fragment are known and well defined. Rather, conjugation randomly
occurs on a plurality of reactive and poorly defined sites. As a
result, also the stoichiometry of the conjugation products, i.e.
the molar ratio diagnostic/therapeutic moiety to protein/protein
fragment, results poorly defined. At best only a mean
stoichiometric molar ratio of conjugating molecule to protein can
be measured and fractional occupancy of certain amino acid residues
can be estimated. The actual final product generally consists of a
complex, poorly defined mixture of variously substituted compounds,
each one having its stoichiometry of substitution. Clinical
application of such mixtures of conjugated products is at variance
with the norms regarding classical pharmaceutical products.
Therefore health regulatory bodies are calling for chemically
better defined conjugates involving immunoglobulins and/or their
fragments.
[0011] Such products, even if highly needed, have so far not been
practical, since by present means they are obtained in low yields
and require costly and industrially impractical separation methods.
It would thus be highly desirable to find a method that allows
conjugation of diagnostic or therapeutic molecules to mAb or its
fragments at a stoichiometric ratio of at least one, and only at
well-defined sites that do not interfere with binding to antigens.
The present invention offers a solution to this problem, preferably
with regard to Fab fragments.
[0012] Fab fragments contain four intra-polypeptide-chain disulfide
bridges and one inter-polypeptide-chain disulfide bridge. The
single inter-chain disulfide bridge is located close to the
carboxy-terminal of the two polypeptide chains, i.e. at the
opposite end of the site on the molecule which is responsible for
antigen binding. Chemical modifications at this site are therefore
expected to have minimal effects on affinity for antigens.
Accordingly, realizing a method able to selectively functionalize
only said inter-chain disulfide bond leaving untouched the other
four intra-chain disulfide bonds, as well as the other possible
reactive groups in the molecule, is of the greatest importance for
obtaining highly pure and structurally well defined compounds.
[0013] Disulfide bridges in proteins can be reduced to pairs of
free sulfhydryl groups. Most often that is accomplished by exposing
the protein to a very large molecular excess of
small-molecular-weight sulfhydryl compounds, such as, for example,
mercaptoethanol, dithiothreitol, dithioerythritol, cysteine or
glutathione. Under these conditions, disulfide bonds are formed
among the small-molecular-weight sulfhydryl compounds, while the
protein disulfide bridges are reduced to free sulfhydryl groups. It
is difficult to eliminate the excess of reducing sulfhydryl
compounds and their oxidation products at the end of the reaction
without causing substantial reoxidation of the protein sulfhydryl
groups to disulfide bridges again. Thus, usually, when a subsequent
sulfhydryl-specific chemical modification of the protein sulfhydryl
groups is desired, the modifying agent is added in the continued
presence of the excess of the small-molecular-weight sulfhydryl
compounds and of their oxidation products. Since all the sulfhydryl
groups in the reaction mixture, both those on the protein and those
on the small-molecular-weight sulfhydryl compounds, undergo the
same reaction with the modifying agent, the latter must be added in
large excess over the number of the protein sulfhydryl groups,
actually somewhat in excess over the sulfhydryl groups of the
small-molecular-weight sulfhydryl compounds. When expensive
modifying agents are at stake, this type of process results
non-practical from the industrial point of view. Moreover, such
conditions are difficult to fully standardise and, worse of all, do
not consent to obtain specific and stoichiometrically well defined
modifications at the desired sulfhydryl group/groups of the protein
or protein fragment of interest, leaving untouched the other ones.
In other words, this type of reaction involves, at least partially,
all the disulfide groups of the protein, and sometimes also other
reactive groups of the same, thus giving a mixture of randomly
reacted and unreacted sulfhydryl groups.
[0014] The present invention mainly focuses on conjugates involving
Fab fragments, substantially because of their size.
[0015] As already mentioned, a lot of literature, also comprising
patents and patent applications, exists, dealing with the
preparation of conjugates of immunoglobulins and their fragments
with suitable diagnostic or therapeutic mojeties. Actually none of
said documents solves, nor gives useful suggestions for solving,
the problem represented by the need of administering to the patient
pharmaceutical formulations containing, as active ingredients, Fab
fragments which have been selectively and quantitatively
functionalized only at desired specific sites of the molecule, thus
showing a predefined, precise substitution stoichiometry.
[0016] EP-A-13 1836, for instance, discloses S-alkylated Fab or Fc
fragments of human immunoglobulins (IgG) obtained by reducing the
multiple inter-chain disulfide bonds with excess of
mercaptoethanol, dithiothreitol or dithioerythritol followed by
alkylation of the resulting sulfhydryl groups. However, the
disclosed method does not allow a precise control of the
stoichiometry of the conjugation on the antibody fragments, thus
giving a complex mixture of the various possible products.
Moreover, the excess of reducing agent makes the use of a large
excess of alkylating agent necessary.
[0017] A similar method is disclosed in U.S. Pat. No. 5,612,016 and
is applied only to intact IgG or to F(ab').sub.2 fragments.
Reduction of the disulfides is performed with excess of thiol
derivatives and can involve more than one disulfide group. The
final conjugate may contain at least one ligand per antibody or
F(ab').sub.2 fragment, but different ratios are equally allowed. No
mention to a precise, well defined conjugation stoichiometry, as
well as to a precise site of conjugation is reported. Also in this
case large excess of alkylating agent is required to overcome the
excess of reducing agent.
[0018] U.S. Pat. No. 5,274,119 shows that selective reduction of
inter-chain disulfide bridges of F(ab').sub.2 fragments with
dithiothreitol is possible only under very strictly controlled
conditions, e.g. at pH=7. Moreover subsequent purification over a
GF-250 HPLC column is mandatory. That means that the method is not
applicable on industrial scale and that at least some of the free
sulfhydryl groups are reconverted to disulfide bonds.
[0019] Other conjugates of antibodies or fragments thereof,
obtained by reaction of the free sulfhydryl groups, deriving from
the reduction of disulfide bridges, with different modifiers of the
sulfhydryl groups, are disclosed in EP-A-453082, U.S. Pat. No.
4,741,900, EP-A-417927, EP-A-023779, EP-A-332022, EP-A-453082,
EP-A-277088, U.S. Pat. No. 5,082,930. However, all these documents
generally disclose chemistries which produce mixtures of products
of non-defined structure; none of them allows or discloses or,
directly or indirectly, teaches the preparation of specifically
substituted conjugates characterized by a pre-determined and
substantially controlled conjugation stoichiometry.
[0020] Very recently, in Bioconjugate Chem, 2001, 12, 178-185, Fab
fragments were described in which the reduction of the disulfide
bonds was performed by using 2-mercaptoethanol. An illustration of
a Fab modified on a C-terminal sulfhydryl group was shown. However,
the described reduction introduced 3.67 thiol groups per molecule
of Fab, thus leading, even in this case, to conjugates of
ill-defined conjugation stoichiometry. It further confirms that the
use of thiol derivatives as reducing agents does not represent the
solution to the need of obtaining the desired selective reduction
of Fab fragments and the corresponding stoichiometrically well
defined conjugation.
[0021] In the case of Fab fragments, which bear the antigen-binding
sites, the problem of selective conjugation at only the C-terminal
sulfhydryl groups is intensely perceived by researchers, but cannot
be addressed by the methods used for intact immunoglobulins.
Despite of this evident need, no other methods have been so far
described that are able to specifically and quantitatively direct
the conjugation reaction only to the sulfhydryl groups which do not
take part in stabilizing the folding of the polypeptide chain, i.e.
the two deriving from the inter-chain disulfide bond.
[0022] The use of particular phosphine derivatives, such as
tributylphosphine is or tris-(carboxyethyl)phosphine (TCEP), as
reducing agents for the disulfide bonds in proteins has already
been disclosed in a number of papers: for example in Methods in
Enzymol. 1977, 47, 116-122, J. Org. Chem. 1991, 56, 2648-2650; Eur.
J. Nucl. Med. 1995, 22, 690-698, Biophisical Journal 1998, 74,
A179, abstr.Tu-Posl96, Faseb Journal 1997, 20 11, A1361, abstr.
2948, Eur. J. Nucl. Med. 1999, 26, 1265-1273, Anal. Biochem. 1999,
273, 73-80; Protein Science, 1993, 2, 1749-1755; nevertheless, said
phosphine agents have never been suggested as possible selective
reducing agents for the inter-chain disulfide bonds in Fab and Fab'
fragments.
[0023] Despite the general teaching of the art, we have now
surprisingly found that it is possible to prepare, easily and with
convenient yields, conjugates of Fab fragments with diagnostic or
therapeutic agents, or useful precursors thereof, which are
characterized, within narrow limits of error, by exact conjugation
stoichiometries, i.e. showing a conjugation molar ratio
agent/agents to Fab of 1:1 or 2:1, being the Fab selectively
functionalized only at one or two specific sulfhydryl groups in a
predefined position of the Fab, i.e. those deriving from the
selective reduction of the disulfide inter-chain bond.
SUMMARY OF THE INVENTION
[0024] In a first preferred embodiment, the present invention
provides a chemical conjugate between an immunoglobulin Fab
fragment and molecular entities imparting diagnostic or therapeutic
utility, whereby the only sites of conjugation on the Fab fragment
are one or both of the sulfhydryl groups deriving from the
selective and quantitative reduction of the inter-chain disulfide
bond of said Fab fragment and whereby said molecular entities
imparting diagnostic or therapeutic utility have at least one free
sulfhydryl-reactive group, characterized in that the conjugation
stoichiometric molar ratio molecular entity to Fab fragment is in
the range from 0.95 to 1.05 or in the range from 1.95 to 2.05.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The conjugate is obtained by selectively and quantitatively
reducing only the inter-chain disulfide bond of a Fab fragment and
then quantitatively functionalizing one of the two obtained
sulfhydryl groups by reaction with a first molecular entity which
has at least one free sulfhydryl-reactive group and gives
therapeutic or diagnostic utility, then, if desired, quantitatively
functionalizing also the other sulfhydryl group of the Fab with a
second molecular entity having at least one free
sulfhydryl-reactive group and imparting diagnostic or therapeutic
utility, said second moiety being identical to the first one or
even different, in this case possibly giving also different
diagnostic or therapeutic properties.
[0026] Alternatively and preferably, after reduction of the
inter-chain disulfide bond, it is possible to quantitatively obtain
the symmetrically diconjugated product by directly reacting said
reduced Fab fragment with a stoichiometric excess of one of said
conjugating moieties.
[0027] The term "quantitatively functionalizing", as used in this
disclosure, means that the final conjugated compounds show:
[0028] a) a molar ratio between conjugating molecular entity and
Fab fragment ranging from 0.95 to 1.05, when only one of the two
free to sulfhydryl groups of the reduced Fab is conjugated,
[0029] b) a molar ratio between conjugating molecular entity and
Fab fragment ranging from 1.95 to 2.05, when both of the two
sulfhydryl groups are conjugated either asymmetrically or
symmetrically.
[0030] In case only one of the sulfhydryl groups deriving from the
selective and quantitative reduction of the inter-chain disulfide
bond of the Fab is desired as a conjugated, the other one
sulfhydryl group may be kept as a free sulfhydryl group or, in
turn, may be functionalized with a bocking group. This blocking
group preferably comprises a chemical moiety non-imparting
diagnostic or therapeutic utility, being said chemical moiety
preferably selected among protective groups of the thiol group or
small alkylating or arylating agents.
[0031] Without thereby limiting the generality of the invention,
preferred examples of first molecular entities having a
sulfhydryl-reactive group and imparting diagnostic or therapeutic
utility comprise suitable derivatives of chelating agents for, or
chelates of, radionuclides, paramagnetic metal ions or luminescent
metal ions, a chromophoric fluorescent or a phosphorescent
molecule, a biotin molecule, a hapten recognized by a distinct
antibody or fragment thereof, an avidin or streptavidin molecule, a
therapeutic drug, a lipophilic chain bearing molecular entity
incorporated into liposomes, phospholipid-stabilized microbubbles,
triglyceride- or polymer-based microspheres, microballoons which
carry the diagnostic or therapeutic agent. Said first moiety may
further comprise one or more functional groups which may be used,
as such or after deprotection or after chemical modification, as
targets for the selective attachment of a second Fab fragment,
equal or different from the first one, or of a second molecular
entity imparting diagnostic or therapeutic utility.
[0032] Without thereby limiting the generality of the invention,
preferred examples of suitable sulfhydryl-reactive groups comprise
iodoacetyl, bromoacetyl, vinyl or maleimido groups, or
polyfluorobenzene or dinitrofluorobenzene derivatives. If desired,
a reversible linkage can be obtained by reaction with another
disulfide-containing molecule and formation of mixed
disulfides.
[0033] The second molecular entity can be the same as the first one
or it may be different, thus giving a combination of different
residues and, possibly, of different diagnostic or therapeutic
effects or even of a mixed diagnostic and therapeutic use.
[0034] Preferred examples of said second molecular entity having a
sulfhydryl-reactive group and imparting diagnostic or therapeutic
utility comprise suitable derivatives of chelating agents for, or
chelates of, radionuclides, paramagnetic metal ions or luminescent
metal ions, a chromophoric fluorescent or a phosphorescent
molecule, a biotin molecule, a hapten recognized by a distinct
antibody or fragment thereof, an avidin or streptavidin molecule, a
therapeutic drug, a lipophilic chain bearing molecular entity
incorporated into liposomes, phospholipid-stabilized microbubbles,
triglyceride- or polymer-based microspheres, microballoons which
carry the diagnostic or therapeutic agent. Said second moiety may
further comprise one or more functional groups which may be used,
as such or after deprotection or after chemical modification, as
targets for the selective attachment of a second Fab fragment,
equal or different from the first one, or of a second molecular
entity imparting diagnostic or therapeutic utility.
[0035] Even in this case preferred examples of suitable
sulfhydryl-reactive groups comprise iodoacetyl, bromoacetyl, vinyl
or maleimido groups, or polyfluorobenzene or dinitrofluorobenzene
derivatives. If desired, a reversible linkage can be obtained by
reaction with another disulfide-containing molecule and formation
of mixed disulfides.
[0036] Fab fragments are obtained by known methods: the use of
rFab, i.e. Fab obtained through recombinant DNA techniques, is
particularly preferred.
[0037] According to another preferred embodiment, the invention
provides a process for the preparation of said conjugates, said
process comprising:
[0038] a) the selective and quantitative reduction of the
inter-chain disulfide bond of a Fab fragment to give two free
sulfhydryl groups;
[0039] b) the quantitative functionalization of one or both of the
sulfhydryl groups from step a) with molecular entities having at
least one free sulfhydryl-reactive group and imparting diagnostic
or therapeutic utility, to give mono- or diconjugate compounds,
said diconjugates deriving from either symmetric or asymmetric
functionalization of the sulfhydryl groups.
[0040] As above disclosed, a number of reducing agents are known
which can be employed for the reduction of disulfide bonds, but, in
the present case, specific reagents and specific reaction
conditions were needed in order to quantitatively reduce only the
inter-chain disulfide bond of a Fab fragment, leaving the other
disulfide bonds unaffected. Namely, it is well known in the art
that reducing agents for the disulfide bond may be selected from
borohydrides, cyanoborohydrides, phosphines, thiol compounds,
stannous ions, ascorbate and dithionite. However, none of them has
been till now disclosed as a specific reducing agent for the
inter-chain disulfide bond od a Fab fragment.
[0041] Unexpectedly, phosphines resulted highly promising for
reaching this scope, in particular tributylphosphine and
tris-(carboxyethyl)phosphine. The last one, hereinafter shortly
named with the acronym TCEP, resulted the reducing agent of choice,
surprisingly allowing to obtain the desired quantitative and
selective reduction only of the inter-chain disulfide bond in Fab
fragments, while leaving unaffected the other four --S--S--
intra-chain bonds. This goal was obtained by using controlled
working conditions and a substantially lower excess of the reducing
agent in comparison to other possible reducing compounds. Moreover,
no interactions usually happened with the conjugating moieties, so
it was also possible to limit the excess of the same during the
following condensation step. As a result, less reactants were used,
less by-products were formed, no need for intermediate purification
of the reduced Fab fragments existed, higher yields of purer,
easier to purify, final compounds were obtained.
[0042] The preferred found experimental conditions under which the
selective and quantitative reduction of the invention takes place
are shortly sunmarized in the following and further detailed in the
experimental section.
[0043] After mixing the reacting species under buffered conditions
(every type of buffer giving the desired pH range is equally
usable) according to the teaching of Examples 1 and 3, a final
buffered aqueous reaction solution having the following
characteristics is obtained:
[0044] Fab concentration: 1-100 .mu.M, preferably 1.5-10 .mu.M,
most preferably 2-5 .mu.M;
[0045] Phosphine concentration: 0.1-10 mM, preferably 0.5-5 mM;
[0046] pH of the buffered solution: between 4 and 8, preferably
between 5 and 7.
[0047] Reaction time ranges from 5 to 180 min, preferably from 25
to 70 mm.
[0048] Reaction temperature is kept from 4 to 45.degree. C.,
preferably from 25 to 40.degree. C.
[0049] The condensation reaction is usually performed immediately
at the end of the reduction of the disulfide bond, in the same
reaction medium, by adding a buffered aqueous solution of the
desired conjugating molecular entity, without previously purifying
the reduced Fab fragment.
[0050] The preferred found condensation conditions are disclosed in
detail in the experimental section, Examples 1, 4 and 6. The final
buffered aqueous reaction solution (every type of buffer giving the
desired pH range is equally usable) preferably has the following
characteristics:
[0051] Fab concentration: 2-5 .mu.M;
[0052] Phosphine concentration: 0.5-5 mM;
[0053] conjugating moiety concentration: 0.1-100 mM;
[0054] pH of the buffered solution: between 5 and 7.
[0055] Reaction time is preferably .gtoreq.30 min.
[0056] Reaction temperature is kept from 4 to 45.degree. C.,
preferably from 20 to 40.degree. C.
[0057] For the purpose of confirming the nature of the conjugates
of the present invention, in particular the stoichiometry of the
condensation reaction, we have conjugated a recombinant anti-Herpes
simplex virus Fab fragment (prepared according to: Cattani P,
Rossolini G M, Cresti S, Santangelo R, Burton D R, Williamson R A,
Sanna P P, Fadda G; J Clin Microbiol. 1997 June; 35(6): 1504-9.
"Detection and Typing of Herpes Simplex Viruses by Using
Recombinant Immunoglobulin Fragments Produced in Bacteria"),
selectively reduced at the inter-chain disulfide bond by using the
method of the invention, with .beta.-maleimidopropionic acid, as
disclosed in Example 1. This last molecule adds a carboxylate
residue to the free sulfhydryl group and thus enables the
measurement of the number and type of conjugated molecules by using
a simple ion-exchange chromatography method.
[0058] The alkylation reaction was performed immediately at the end
of the reduction of the disulfide bond, in the same reaction
medium, without purifying the reduced Fab fragment.
[0059] Said alkylation reaction was performed under the preferred
found conditions of the invention as fully disclosed in Example
1.
[0060] At the end of the reaction, it was possible to calculate the
total number of added carboxylate groups, in this case confirming
that both of the two free sulfhydryl group underwent the
conjugation reaction, as also. shown in FIG. 1.
[0061] The reaction conditions may vary according to the reactivity
of the various thiol-reactive molecular entities, to their
molecular weight and steric hindrance, to the desired final
compound (mono- or di-conjugated, symmetrically or not) and it is
generally advisable to control that, if the reduction step is
omitted, no lateral reaction occurs (this confirms that only the
two sulfhydryl groups deriving from the reduction of the single
inter-chain disulfide bridge of the Fab react with the conjugating
moiety/ies).
[0062] It is particularly preferable to perform the alkylation
reaction without previous separation of the excess of reducing
agent, because the inter-chain disulfide bond can easily
reform.
[0063] The conjugate compounds of the invention are particularly
advantageous because: [0064] a pre-determined, controlled
stoichiometry of conjugation greatly reduces the percentage of
residual impurities, which can be inactive or inhibitory, or even
toxic, in the final compound; [0065] the products are easily
characterised and characterizable for drug registration purposes
before the health authorities; [0066] the process for the
preparation of the products is relatively easy, has good yields and
is applicable on industrial scale; [0067] the purification of the
final diagnostic or therapeutic compounds, or of their precursors,
results simple because it implies the separation of mixtures mainly
containing products with 0, 1 or 2 substituents, said mixtures
being greatly enriched in only one of them; [0068] the conjugated
moieties are exclusively located near the carboxy terminal of the
Fab heavy and light chains, therefore they are not likely to
interfere with the antigen recognition site, which is formed by
residues near the amino terminal part of the polypeptide chains;
[0069] the initial conformation of the Fab is maintained.
[0070] Fab conjugates according to the invention will usually be
directed 25 against antigens of therapeutic or diagnostic interest,
e.g. against tumor antigens, receptors, tissue markers, markers for
specific pathologies, infections, inflammations, degenerative
processes and so on.
[0071] So, according to a further preferred embodiment, the
invention also provides diagnostic and/or therapeutic compositions
containing said conjugates as active ingredients.
[0072] For the desired diagnostic or therapeutic applications, the
conjugate compounds of the invention will be formulated in suitable
compositions, usually in the form of suspensions, solutions or
emulsions for parenteral administration, lyophilizates to be
reconstituted before use or even in the form of other
pharmaceutical compositions suitable for other desired different
types of administration. The dose will depend on several parameters
( kind of ligand, patient's conditions) but it will generally be in
the range from 0.1 to 10 mg of conjugate per single administration
in the case of diagnostic applications and in the range from 10 to
500 mg of conjugate per single administration in the case of
therapeutic applications.
[0073] The conjugate compounds of the invention are particularly
advantageous also for their in vitro use, whereby they show their
utility, preferably when applied to immunochemical tests in
vitro.
[0074] The invention equally applies to Fab' fragments, which, as
previously mentioned, have structure similar to and dimension not
much larger than Fab.
EXPLANATION OF THE FIGURES
[0075] FIG. 1 shows cation-exchange HPLC analyses of reaction
mixtures between the rFab of Example 1 and
.beta.-maleimidopropionic acid. In all runs, the peaks eluting
before 5 min are due to salts and reactants that absorb at 215 nm.
[0076] A) is the complete reaction mixture, containing both TCEP
and .beta.-maleimidopropionic acid; [0077] B) is the initial
unreacted rFab solution; [0078] C) is the incomplete reaction
mixture, without TCEP, but containing .beta.-maleimidopropionic
acid.
[0079] A) shows that the initial rFab has completely reacted,
giving a unique conjugation compound. Mass Spectrometry (MS)
analysis demonstrated that the disubstituded product was obtained.
Mass Spectrometry used was of the type MALDI-TOF-MS
(Matrix-Assisted-Laser-Desorption-Ionozation Time-Of-Flight Mass
Spectrometry).
[0080] B) shows the initial rFab of Example 1, which comprises the
rFab and a minor component/impurity consisting of a monodeamidated
rFab;
[0081] C) shows that the reaction is specific and does not take
place in absence of the reducing agent.
[0082] FIG. 2 shows HPLC cation-exchange analysis of the rFab
essentially free from deamidated form, before (up) and after
(bottom) exhaustive conjugation with Compound D of Example 2,
corresponding to lanes 3 and 4 of FIG. 3. The profiles show that
the main peak shifts to lower retention times, due to conjugation,
and that the purity of the preparation, once removed the reagents,
is similar to that of the starting rFab (the peaks at the void
volume around 3 min are due to reagents).
[0083] FIG. 3 shows native electrophoresis of rFab preparations,
before and after exhaustive alkylation with Compound D of Example
2. The reaction mixtures were analyzed without any purification
step. The reduction in migration distance that follows reaction
with Compound D confirms the attachment of a definite number of
negatively charged groups. MALDI-TPOF MS analysis confirmed that
the disubstituted product was obtained.
[0084] 1. rFab (prep. 1)
[0085] 2. rFab (prep. 1) conjugated with Compound D
[0086] 3. rFab (prep. 2)
[0087] 4. rFab (prep. 2) conjugated with Compound D
[0088] 5. rFab (prep. 3)
[0089] 6. rFab (prep. 3) conjugated with Compound D
[0090] The invention is further illustrated in details by reference
to the following non-limiting Examples:
EXAMPLE 1
[0091] Reduction and alkylation of a recombinant anti-Herpes
simplex virus Fab with .beta.-maleimidopropionic acid.
[0092] A model reaction system was established in order to test
several reaction conditions and to easily characterize the reaction
products. A commercially available maleimido derivative endowed
with a ionizable group, .beta.-maleimidopropionic acid (following
compound of formula I), was
[0093] selected as a model compound, which allowed the evaluation
of the number of conjugated moieties by a simple ion-exchange
chromatography
[0094] analysis, together with MS analysis ##STR1##
[0095] The optimised procedure was the following one:
[0096] One volume V of a 2 mM TCEP solution was prepared by 1 to
250 dilution of the 0.5 M commercial product (Pierce) in a
thoroughly deareated buffer containing 50 mM Tris-HCl, 5 mM EDTA at
pH=7.0. Then, this solution was added to an equivalent volume V of
a 10 .mu.M solution of the rFab of the title (prepared according to
the previously mentioned Cattani P et al. reference) and incubated
for 30 min at 37.degree. C.
[0097] Then, a V/2 volume of 50 mM .beta.-maleimidopropionic acid
in 0.1 M acetate buffer at pH=5 was added and the reaction mixture
was kept 1 h at 37.degree. C. At this point the reaction is
complete and, if required, excess reactants can be removed by
conventional separation procedures, like dialysis or
gel-filtration.
[0098] For analytical purposes, a sample was injected into a
cation-exchange HPLC column and eluted with a salt gradient.
Chromatography was performed on a WP Carboxy-sulfon column (J. T.
Baker) at 1 mL/min, using a 15-min gradient from 60 to 120 mM
phosphate buffer pH 5.8. Detection was performed at 215 nm. The
results are shown in FIG. 1 and demonstrate that the rFab is
completely converted in a homogeneous product having 2 more
negative charges (FIG. 1A), therefore corresponding to the
disubstituted derivative. MS analysis confirmed the disubstitution.
The same initial rFab, unconjugated, is shown in FIG. 1B, together
with a small peak eluting at 8.8 min, which is due to a
mono-deamidated form of the rFab and is a useful marker of the
elution position of rFab species differing only by 1 charge. Also
this species is subjected to reduction and alkylation and gives the
corresponding disubstituted derivative eluting at 6.38 min in FIG.
1A. FIG. 1C shows the content of a reaction mixture in which the
reduction step was omitted, i.e. the reducing agent TCEP was not
added. The unmodified elution profile, with respect to the
unconjugated rFab of FIG. 1B, demonstrates that the alkylation is
specific for thiol groups and does not take place if disulfide
bonds are intact.
[0099] The rFab of Example 1, after selective reduction of the
inter-chain disulfide bond, was reacted with a novel maleimido
derivative of diethylentriaminopentaacetic acid (DTPA), which is a
well known and widely used chelating agent of proven diagnostic and
therapeutic utility (Compound D of Example 2), to give the
conjugation products.
EXAMPLE 2
Synthesis of
N.sup.2,N.sup.2-bis[2-[bis(carboxymethyl)amino]ethyl]-N.sup.6-[4-(2,5-dio-
xo-1H-pyrrol-1-yl)-1-oxobutyl]-L-lysine (Compound D).
[0100] The compound of the title was synthesised starting from
compound A (which was prepared according to "Anelli, P. L. et al.;
Bioconjugate Chem. 1999, 10, 137-140") following the two steps
scheme below:
[0101] First Step:
[0102] Isobutyl chloroformate (15 mmol) was dropped into a solution
of 4-maleimidobutyric acid of commercial source (Compound B; 13.6
mmol) and triethylamine (15 mmol) in tetrahydrofuran (55 mL) at
-15.degree. C., under nitrogen atmosphere. After 15 min, a solution
of compound A (13.6 mmol), prepared as previously disclosed, in
tetrahydrofuran (20 mL) was dropped therein, while keeping the
temperature at 4.degree. C. After 15 min cooling was interrupted
and the mixture was stirred at room temperature for 1 h, then
evaporated under vacuum. The residue was dissolved in ethyl acetate
(50 mL), then washed with water. The organic phase was then dried
over sodium sulphate, filtered and evaporated under vacuum.
[0103] The residue was purified by flash chromatography in an ethyl
acetate/petroleum ether mixture, to obtain Compound C as a yellow
oil (yield: 8.12 mmol, equivalent to 60%). The analytical data are
consistent with the desired structure.
[0104] Second Step:
[0105] Trifluoroacetic acid (68.6 mmol) was added to a solution of
Compound C (6.25 g, 6.86 mmol) in dichloromethane (100 mL). After
15 h the solution was evaporated under vacuum and the residue was
taken up into a further 10 ml of trifluoroacetic acid. After 6 h,
the mixture was ##STR2##
[0106] evaporated again, the residue was taken up into 50 mL of
water, purified on an Amberlite XAD 16.00 T column with a
water/acetonitrile mixture and the relevant fractions were
evaporated, to obtain Compound D as a white solid (59% yield). The
analytical data are consistent with the suggested structure
EXAMPLE 3
[0107] Selective Reduction of the rFab Inter-Chain Disulfide
[0108] Reduction of one volume V of the 10 .mu.M rFab solution of
Example 1 was carried out with an equivalent volume V of a 5 mM
TCEP solution in 100 mM acetate buffer at pH=5, for 1 h at
37.degree. C. As much oxygen as possible was removed from the
reaction medium by bubbling nitrogen through the buffering agent
before use. Under these conditions, the reduction of the
inter-chain disulfide was substantially complete, as observed by
SDS-PAGE analysis. However, rFab conformation was not lost as
evidenced by the fact that, removing the reducing agent and
incubating the reduced rFab for 2 h in 0.1 M Tris-HCl at pH=8, the
inter-chain disulfide was formed again. This fact is very
important, meaning that the final product will maintain the
capability of recognising the reactive site of the antigen.
EXAMPLE 4
[0109] Diconjugation of Compound D with the Reduced rFab.
[0110] The cysteines formed as a consequence of the specific
reduction of Example 3 were subjected to conjugation with Compound
D od Example 2, directly in the same reaction medium of the
reduction, by simply adding 25 a half volume V/2 of a 100 mM
Compound D solution in 0.5 M sodium acetate (final pH of the
reaction solution of about 5) and incubating for 16 h at 30.degree.
C. Under the described conditions, a single product formed in
quantitative yield as shown in FIG. 2, where HPLC cation-exchange
analysis of rFab before (up) and after (bottom) conjugation with
Compound D is reported.
[0111] Chromatography was performed on a WP Carboxy-sulfon column
(J. T. Baker) at 1 mL/min, using a 15-min gradient from 60 to 120
mM phosphate buffer pH 5.8. Detection was performed at 215 nm.
[0112] MS analysis of the final product confirmed the formation of
the diconjugate.
EXAMPLE 5
[0113] Characterisation of the Diconjugate of the Reduced rFab with
Compound D.
[0114] As the introduction of each DTPA derivative molecule causes
an increase in the protein total negative charge, protein charge
analysis may be used to evaluate the homogeneity of the
preparation, i.e. to make sure that no conjugates with a variable
number of DTPA derivatives are formed, thus leading to a mixture of
different compounds. Charge analysis can be carried out by
electrophoretic and chromatographic techniques. The used
electrophoretic technique was native electrophoresis. According to
this technique, protein migration depends on both molecular weight
and charge, however, for the reduced rFab before and after
conjugation with Compound C, being the protein mass substantially
the same, the difference of the electrophoretic run was due solely
to the differences in the charge introduced by the DTPA derivative.
As shown in FIG. 3, three preparations of rFab analysed before and
after conjugation with Compound D showed the same behaviour, i.e. a
reduction of the migration distance towards the cathode, following
conjugation. The same analysis showed that the conjugation product
was homogeneous and reproducible.
EXAMPLE 6
[0115] Monoconjugation of the Reduced rFab with Compound D.
[0116] The inter-chain disulfide bond of the rFab of Example 1 (one
volume V of the 10 .mu.M rFab solution) was selectively reduced as
described in Example 3, then a half volume V/2 of a 0.5 mM Compound
D solution in 0.5 M sodium acetate (final pH of the reaction
solution of about 5) was very slowly dropped directly into the same
reaction medium of the reduction at about 5.degree. C. and the
condensation was continuously monitored by HPLC.
[0117] The formation of the product of mono-alkylation was shown by
the appearance of an increasing peak of intermediate retention time
between the unreacted reduced rFab and the diconjugation product.
When the area of this peak became bigger than the ones of the other
two products, the reaction was stopped and the final mixture was
purified by chromatography. MS analysis confirmed that the main
peak corresponded to the monoalkylated compound.
* * * * *